Method of Improving Resistance to Substrate Analog of Nitric Acid in Microalga

Abstract

A method of improving resistance to a substrate analog of nitric acid in a microalga, containing

deleting a gene encoding the following protein (A) or (B) present in the genome of the microalga, or downregulating expression of a gene encoding the following protein (A) or (B):

(A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 41; and

(B) a protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence of the protein (A), and having nitrate transporter activity.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of improving resistance to a substrate analog of nitric acid in a microalga, a transformant of a microalga having resistance to a substrate analog of nitric acid, and a method of preparing a transformant having resistance to a substrate analog of nitric acid.BACKGROUND ART[0002]Culture of microorganisms for producing a biofuel or other useful substance is generally performed on the assumption of culturing pure culture of target microorganisms. Accordingly, in order to prevent contamination by microorganisms other than the desired microorganisms, sterilization treatment or germicidal treatment is usually applied to the culture medium or incubator.[0003]However, as scale of microorganism cultivation is expanded, the cost of energy and equipment required for the sterilization or germicidal treatment of the culture medium and the like increases. Further, even if culture medium subjected to sterilization or germicidal treatme...

AI Technical Summary

Benefits of technology

[0018](D) a protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence of the protein (C), and having nitrate reductase activity (hereinafter, also merely referred to as “NR activity”).

[0019]Further, the present invention relates to a transformant of a microalga having resistance to a substrate analog of nitric acid, wherein a gene encoding the protein (A) or (B) present in the genome is deleted, or expression of a gene encoding the protein (A) or (B) is downregulated.

[0020]Further, the present invention relates to a transformant of a microalga having resistance to a substrate analog of nitric acid, wherein a gene is deleted or gene expression is downregulated for each a gene encoding the protein (A) or (B) and a gene encoding the protein (C) or (D) present in the genome.

[0021]Further, the present invention relates to a method of preparing a transformant having resistance to a substrate analog of nitric acid by deleting a gene encoding the protein (A) or (B) present in the genome of a microalga, or downregulating expression of a gene encoding the protein (A) or (B), thereby obtaining the transformant using resistance to the substrate analog of nitric acid as an indicator.

[0022]Furthermore the present invention relates to a method of preparing a transformant having resistance to a substrate analog of nitric acid by deleting a gene or downregulating gene expression for each a gene encoding the protein (A) or (B) and a gene encoding the protein (C) or (D) present in the genome of a microalga, thereby obtaining the transformant using resistance to the substrate analog of nitric acid as an indicator.

[0023]Other and further objects, features and advantages of the invention will appear more fully from the following description, appropriately referring to the accompanying drawings.

Problems solved by technology

However, as scale of microorganism cultivation is expanded, the cost of energy and equipment required for the sterilization or germicidal treatment of the culture medium and the like increases.

Further, even if culture medium subjected to sterilization or germicidal treatment is used, growth of non-targeted microorganisms owing to contamination arising after the treatment is hard to avoid.

When microorganisms (typically microalgae) are cultured in an open area using an open-type open pond or the like, risk of contamination is particularly high.

However, a strain prepared by introducing a foreign gene corresponds to a gene recombinant, and use thereof is restricted under the regulatory requirements of the Cartagena Protocol, for example, owing to its risk of spreading the foreign gene across the natural environment.

Further, the microalgae attract attention as next-generation biomass resources, because the microalgae do not compete with foods.

However, nothing has been reported regarding use of endogenous gene of microalgae in the class Eustigmatophyceae to impart drug resistance.

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